Blood coagulation is a complex system involving a large number of proteins that function in concert to yield hemostasis. The coagulation system is regulated by a series of proteins present in plasma and on the surface of cells. Under physiological conditions, pro- and anti-coagulant mechanisms are delicately balanced to provide hemostasis and coagulation. Disturbances in this balance result in either bleeding or thromboembolic disorders, and can be induced by medical conditions, congenital or acquired, the intake of drugs or vitamins. The most widely used tests to measure the coagulation status of a person are known as clotting assays. Clotting assays measure the clotting times of recalcified platelet-poor plasma. The prothrombin time (PT) test is performed by adding calcium and thromboplastin to citrated plasma. The term thromboplastin refers to a phospholipid-protein extract of tissues, usually lung, brain, or placenta, that contains both tissue factor and the phospholipid necessary to promote the activation of factor X by Factor VII. The standard thromboplastin is xe2x80x9cThe World Health Organizationxe2x80x9d (WHO) thromboplastin which is derived from human brain thromboplastin. PT test instruments are based upon measuring how the plasma viscosity changes over time after calcium and thromboplastin have been added to plasma. When plasma coagulates it turns from a solution to a gel which is more viscous. In the APTT test, decalcified plasma is incubated for a specified time with activating agents such as ellagic acid, silica, soy extract, or kaolin. Following incubation with the activating substance, calcium chloride is added to the plasma mixture for a clot to form. In the PTT test, plasma is incubated for 3 minutes with a reagent supplying procoagulant phospholipid and surface-active powder (e.g. micronized silica). Calcium is then added and the clotting time noted.
Although widely used, clotting assays present several drawbacks. First, clotting assays are by definition invasive, as they require some blood to be drawn from the patient. Second, they require that actual coagulation of the blood be performed in a vial, which involves delicate handling, accurate timing, and citration of plasma. Third, clotting assays depend on an additional product, thromboplastin, whose performance can be different from the reference WHO thromboplastin, requiring complicated International Normalization Ratio (INR) calculations to ensure accuracy. Finally, clotting tests are indirect tests, reflecting only indirectly fibrinopeptides and fibrinogen degradation products and other proteins related to the state of anticoagulation.
Direct measurement of prothrombin fragments in blood or serum for determining the coagulation status is known in the art (U.S. Pat. No. 5,071,954). However, these methods are invariably invasive since they require some blood to be drawn.
Measurements of at least some of the above fragments in urine may be found in various papers, for instance in A. Bezeaud and al, Thrombosis Research, 13, 3, p. 551-556 (September 1978); J. V. Sorensen and al, Thrombosis Research, 67, 4, p. 429-434 (August 1992); D. M. Weinstock and al., American J. Hematology, 57,3, p. 193-199 (March 1998).
The purpose of this invention is to use a non invasive diagnostic test technique to measure the level of blood anticoagulation induced by pharmaceuticals which prevent or reduce coagulation of blood as well as to measure and/or identify any natural or disease-induced blood disorders which effect the coagulation of blood. In addition, the invention can be used to measure the level of hypercoagulation induced by drugs, proteins and vitamins which augment coagulation of blood (procoagulant drugs). The invention is a non invasive test since it measures key substances present in saliva, filtered saliva, sputum, or the like, which are obtainable non invasively (hereunder referred to as xe2x80x9csalivaxe2x80x9d). The invention is usable in humans as well as in animals.
It must be pointed out at this stage that saliva and urine are very different in terms or biological roles and compositions, as well as in their enzymatic environments.
Examples of clinical applications in which the test could be used include all blood coagulation disorders, be they congenital, acquired, or drug-induced. One can mention congenital deficiencies of the intrinsic pathway of the coagulation system (hemophilia A and B, deficiencies of Fitzgerald factor and Fletcher factor), deficiencies in protein C and protein S, heparin and heparin-like therapy, Warfarin (Coumadin)-like therapy, acute thrombotic situations such as acute myocardial infarction or pulmonary embolism, Vitamin K deficiency or excess, hypofibrinogenemia, liver disease, disseminated intravascular coagulopathy, among others.
The invention produces a quantitative measurement of prothrombin fragment 1+2 (F1+2), prothrombin fragment 1 (F1), prothrombin fragment 2 (F2), fibrinopeptide A (FpA) and D-Dimers in saliva which is correlated with the coagulation time as expressed for example by prothrombin time (PT), INR, partial thromboplastin time (PTT) and activated partial thromboplastin time (APTT). Fragments F1+2, F1, F2, FpA and D-Dimers are preferably measured by means of immunoassay techniques, which measure a substance using the reaction of an antibody (immunoreagent) with an antigen (i.e. the protein to be measured). The quantification of the substance measured is determined by measuring how much of the antigen is bound to the antibody and how much of the antigen is not bound to the antibody. Enzyme-labeled, fluorescent-labeled, phosphorescent-labeled, radio-labeled, chemiluminescent-labeled and bioluminescent-labeled immunoassay techniques are for example usable to measure the concentrations of Fragments F1+2, F1, F2, FpA and D-Dimers. Capillary action, precipitation, turbidometric, diffusion, agglutination and electrophorefic immunoassay techniques can also be used in practicing the invention, as well as potentiometric, amperometric, piezoelectric and evanescent-wave immunosensors. In addition, any combination of the aforesaid assay techniques can also be employed to measure the concentrations of Fragments F1+2, F1, F2, FpA and D-Dimers in saliva.
A preferred application of this invention is to control the level of any pharmaceutical which results in anticoagulation of the blood by affecting the conversion of blood Factor X to Factor Xa, which is commonly termed activated Factor X. Factor X and Factor Xa, which are in the latter stages of the coagulation cascade best correlate with the anticoagulation effect (see FIG. 1 for diagram of coagulation cascade).
Factor X is a glycoprotein of molecular weight 55,000 and composed of two polypeptide chains linked by one disulfide bond. The light and heavy chains have molecular weights of 16,000 and 38,000 daltons respectively. During the coagulation process Factor X is converted to Factor Xa by either factors IXa, and VIII or Factor VII and tissue Factor. Factor X can also be activated by other proteases such as trypsin. The activation of Factor X by each of these systems involves the cleavage of a single specific arginyl-isoleucine peptide bond in the heavy chain of Factor X. This gives rise to the formation of a glycoprotein of molecular weight 44,000 and a peptide with a molecular weight of 11,000.
A common feature of Fragments F1+2, F1, F2, FpA and D-Dimers as measured in the invention is that their concentration rises when the conversion of Factor X to Factor Xa increases, and decreases when the conversion of Factor X to Factor Xa is inhibited. The respective roles of Fragments F1+2, F1, F2, FpA and D-Dimers in coagulation is explained in the following paragraphs.
Activated blood factor X forms a -complex with activated blood factor V, phospholipid and calcium. The first step in the activation by the complex is the proteolytic cleavage of a fragment from the NH2-terminal end of prothrombin. This gives rise to what is termed intermediate II (an intermediate precursor of thrombin) and the clipped fragment referred to as prothrombin fragment 1+2. Intermediate II (which is a single chain polypeptide) is then cleaved a second time by activated factor X complex to yield a two chain (light and heavy chains) thrombin molecule.
Thrombin can use prothrombin as a substrate. When thrombin uses prothrombin as a substrate the cleavage pattern differs from that obtained by the activated factor X complex. Thrombin cleaves prothrombin releasing a fragment smaller than that cleaved by the activated factor X complex. This gives rise to what is termed prothrombin fragment 1 and intermediate I. This cleavage occurs without the involvement of activated factor V, phospholipid or calcium. Thrombin cannot activate prothrombin to thrombin. Activated factor X is required for the conversion to thrombin; however, activated factor X then cleaves off the remaining portion of prothrombin fragment 1+2 from intermediate I to give rise to fragment 2 and intermediate II. Intermediate II is then cleaved by activated factor X to give the thrombin molecule. Thrombin generated by the activated factor X mechanism will also cleave the prothrombin fragment 1+2 produced by the initial activated factor X cleavage to give rise to prothrombin fragment 1 and prothrombin fragment 2. See FIG. 2 for diagram of Prothrombin to Thrombin conversion pathway.
The conversion of fibrinogen to fibrin by thrombin initially results in the formation of fibrin 1 monomer and the release of Fibrinopeptide A (FpA). Fibrin 1 monomer is able to polymerize, and thrombin can then cleave the polymer, thereby resulting in the generation of fibrin II and the release of fibrinopeptide B. When Factor Xa is inhibited by a pharmaceutical, the level of FpA will decrease. FIG. 3 shows the generation of FpA by Thrombin.
Fibrin clot Formation is initiated when thrombin cleaves fibrinopeptides A and B from the E domain of fibrinogen. The resulting soluble fibrin monomers align in a staggered end-to-end arrangement to produce protofibrils. Protofibrils later polymerize side-by-side into large, soluble fibrin oligomers. In the process of fibrinogen or fibrin degradation by plasmin within a clot, specific molecular fragments are produced called fibrinogen degradation products (FDP). Plasmin cannot distinguish between fibrinogen and fibrin : therefore, it degrades both. This results in the appearance of essentially the same fragments from fibrinogen and fibrin. FIG. 4 shows the sequence of the reactions in the degradation of fibrinogen and fibrin by plasmin and the principal products, fragment X,Y,D (D-D dimer), and E. Fragments X and Y are referred to as the early degradation products. Fragments D and E are the late degradation products. Fragment X is the first and largest fragment formed (250,000 Daltons). Fragment X is the result of plasmin cleavage of the terminal portion of the alpha chains from a fibrin polymer, leaving isolated fibrin stands. Fragment X is then cleaved by plasmin to form two fragments called Y (YY) and an intermediate complex DXD. This complex is further cleaved into intermediate complexes DED and DYND until finally fragments E and
D (D-D dimer) are formed.
The molecular weight of each fragment is as shown below:
In summary, any pharmaceutical which reduces the conversion of Factor X to Factor Xa will reduce the level of Fragment 1+2, F1, F2, FpA and D-Dimer in body fluids. Reductions in Factor Xa result in blood taking a longer time to coagulate. Therefore, the quantity of Fragment 1+2, F1, F2, FpA and D-Dimer is inversely proportional to the level of anticoagulation. The less Fragment 1+2, F1, F2, FpA and D-Dimer in saliva, the longer it will take for blood to coagulate. Conversely, the more Fragment 1+2, F1, F2, FpA and D-Dimer in saliva, the less time it will take the blood to coagulate.
Both saliva and urine are excreted by the body and both substances are know to contain blood proteins. The advantages and disadvantages of using these medium to diagnose the state of coagulation indicates that saliva would be the preferred medium if the concentration of the coagulation markers in saliva are directly proportional to their concentration in blood.
Compared to urine, saliva presents a number of crucial advantages, for instance:
nearly everyone can salivate at any time of day and for multiple times,
privacy is not required to provide a specimen in contrast to urine specimens,
the extensive vascular bed in the salivary glands offers a good pathway of blood components to the inside of the mouth,
individuals find handling their saliva is more tasteful than handling their urine
equally applicable to men and women, women have blood in their urine during menstruation,
collection of specimens can be fast, so there is less time for deleterious substances in the saliva to effect the concentration of the coagulation marker,
less individuals have salivary gland problems than have kidney problems.
By contrast, disadvantages of saliva over urine are of low importance:
gingival crevicular fluid may contain blood,
saliva flow rate varies due to many factors (for example stress and degree of hydration).
Moreover, the transport of coagulation markers to saliva is a more direct route from blood than urine. The urine is stored in the bladder before excretion and this is a major drawback of urine use as a diagnotic medium.
The invention is relating as well to a non invasive device for doing in-vitro analysis of the concentration of the coagulation markers which best correlate to the state of anticoagulation of individuals undergoing Warfarin therapy so that the individuals can control their Warfarin dosage, thereby reducing visits to care-giver offices and increasing the accuracy of dosing. The functional requirements of the test are: be non invasive; not require a blood sample to be withdrawn from a user; be able to be used at home by persons undergoing Warfarin therapy; be able to be carried out by persons of average intelligence and motor skills after receiving minimal training; results to be readable by the users eye or an inexpensive device such as a photometer, densitometer or calorimeter; be cost effective in that the cost of the test is to be less than the cost charged by physicians and clinics for the patient visit and the cost of the test procedure; be capable of accurately measuring for a person which is stabilized on Warfarin therapy, the coagulation markers over the expected range in the saliva being analyzed; not require meticulous attention to specimen acquisition and processing; only require xe2x80x9cspotxe2x80x9d saliva; portable due to small size and weight; provides rapid results, preferably less than 15 minutes; provides a quantitative result, preferably in the form of a permanent record.
Such an at home test saves time and money, helps those patients which have a hardship traveling to institutions, the debilitated and those lacking adequate transportation.
An at home test also improves anticoagulation therapy and safety as self monitoring has proven more accurate than institution based monitoring, especially for patients interested in their well being. It induces patients to have a greater responsibility for managing one""s own therapy, allows for more frequent monitoring, especially when fluctuating PT values require adjusting of the anticoagulant dose.
Studies conducted of individuals which self monitor and control Warfarin dosage compared to those which are monitored and controlled by a clinic show that the self controlled have less complications and better control. Subjects also would rather self control than spend their time going to a dinic.